U.S. patent application number 13/446712 was filed with the patent office on 2012-10-18 for biocompatible adhesive materials and methods.
This patent application is currently assigned to MASSACHUSETTS INSTITUTE OF TECHNOLOGY. Invention is credited to Natalie Artzi, Elazer R. Edelman, N ria Oliva Jorge, Maria Carcole Solanes.
Application Number | 20120263672 13/446712 |
Document ID | / |
Family ID | 46026933 |
Filed Date | 2012-10-18 |
United States Patent
Application |
20120263672 |
Kind Code |
A1 |
Artzi; Natalie ; et
al. |
October 18, 2012 |
Biocompatible Adhesive Materials and Methods
Abstract
Biocompatible adhesive materials, such as for use with
biological tissues and/or medical implants, are provided, as well
as methods and kits for making and using the biocompatible adhesive
materials. The biocompatible adhesive materials include a dendrimer
component and a polymer component, and may be tailored for specific
tissue types and conditions.
Inventors: |
Artzi; Natalie; (Brookline,
MA) ; Edelman; Elazer R.; (Brookline, MA) ;
Jorge; N ria Oliva; (Barcelona, ES) ; Solanes; Maria
Carcole; (Barcelona, ES) |
Assignee: |
MASSACHUSETTS INSTITUTE OF
TECHNOLOGY
Cambridge
MA
|
Family ID: |
46026933 |
Appl. No.: |
13/446712 |
Filed: |
April 13, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61474984 |
Apr 13, 2011 |
|
|
|
Current U.S.
Class: |
424/78.17 ;
604/82 |
Current CPC
Class: |
A61L 24/08 20130101;
A61L 24/0015 20130101; A61M 5/31596 20130101; A61P 17/02 20180101;
A61L 24/0005 20130101; A61L 24/043 20130101; A61P 29/00 20180101;
A61M 5/19 20130101; A61L 24/08 20130101; C08L 5/02 20130101 |
Class at
Publication: |
424/78.17 ;
604/82 |
International
Class: |
A61K 31/785 20060101
A61K031/785; A61P 17/02 20060101 A61P017/02; A61M 5/31 20060101
A61M005/31; A61P 29/00 20060101 A61P029/00 |
Claims
1. A method for treating, adhering, or sealing biological tissue,
the method comprising: providing a first solution comprising a
polymer component, wherein the polymer component comprises a
polymer having one or more aldehyde groups; providing a second
solution comprising a dendrimer component, wherein the dendrimer
component comprises a dendrimer having at least 2 branches with one
or more surface groups, wherein less than 75% of the surface groups
comprise at least one primary or secondary amine; combining the
first and second solutions together to produce an adhesive
formulation and contacting one or more biological tissues with the
adhesive formulation; and allowing the adhesive formulation to cure
in contact with the one or more biological tissues.
2. The method of claim 1, further comprising the step of adjusting
the concentrations of at least one of the first solution and second
solution to compensate for the characteristics of a biological
tissue.
3. The method of claim 2, wherein the adjusting step comprises
increasing the concentration of the first solution, and
subsequently lowering the concentration until adequate treatment,
adhesion, or sealing is achieved.
4. The method of claim 1, wherein the polymer has a molecular
weight from about 1,000 to about 1,000,000 Da.
5. The method of claim 1, wherein the polymer's degree of
functionalization is from about 10% to about 75%.
6. The method of claim 1, wherein the polymer is a
polysaccharide.
7. The method of claim 6, wherein the polymer is a dextran.
8. The method of claim 7, wherein the dextran has a molecular
weight of about 10 kDa.
9. The method of claim 7, wherein about 50% of the dextran's
hydroxyl groups are aldehydes.
10. The method of claim 1, wherein the dendrimer extends through at
least 2 generations.
11. The method of claim 1, wherein the dendrimer has a molecular
weight of about 1,000 to about 1,000,000 Da.
12. The method of claim 1, wherein the dendrimer is a generation 5
PAMAM-derived dendrimer or a dendrimer derived by oxidizing a
starting dendrimer comprising surface groups comprising at least
one hydroxyl.
13. The method of claim 1, wherein the dendrimer is a generation 5
dendrimer having primary amines on about 25% of the dendrimer's
surface groups.
14. The method of claim 1, wherein the first solution and second
solution are combined on a biological tissue.
15. The method of claim 1, wherein the second solution is a applied
to a tissue followed by the first solution.
16. The method of claim 1, wherein the one or more biological
tissues comprise human tissue.
17. The method of claim 1, wherein the dendrimer component, polymer
component, or both further comprise an additive selected from the
group consisting of foaming agents, pH modifiers, thickeners,
antimicrobial agents, colorants, surfactants, and radio-opaque
agents.
18. The method of claim 1, wherein the first solution is an aqueous
solution.
19. The method of claim 1, wherein the second solution is an
aqueous solution.
20. The method of claim 1, wherein the dendrimer component, polymer
component, or both components, comprise a drug.
21. The method of claim 1, wherein the dendrimer component, polymer
component, or both components, comprise stem cells or other
cells.
22. The method of claim 1, wherein the adhesive formulation reduces
inflammation, enhances healing, or both in the biological
tissue.
23. A kit for making an adhesive comprising: a first part which
includes a first solution comprising a polymer component, wherein
the polymer component comprises a polymer having one or more
aldehyde groups; and a second part which includes a second solution
comprising a dendrimer component, wherein the dendrimer component
comprises a dendrimer having at least 2 branches with one or more
surface groups, wherein less than 75% of the surface groups
comprise at least one primary or secondary amine.
24. The kit of claim 23, further comprising a syringe, wherein the
first and second solutions are stored in the syringe.
25. The kit of claim 24, wherein the syringe comprises separate
reservoirs for the first solution and second solution.
26. The kit of claim 23, wherein the syringe comprises a mixing
tip.
27. The kit of claim 23, further comprising at least two reservoirs
with different concentrations of the first solution.
28. The kit of claim 23, further comprising at least two reservoirs
with different concentrations of the second solution.
29. The kit of claim 23, further comprising instructions for
selecting an appropriate concentration of at least one of the first
solution or second solution to compensate for the characteristics
of one or more biological tissues.
30. The kit of claim 23, wherein the dendrimer component, polymer
component, or both components, comprise a drug.
31. The kit of claim 23, wherein the dendrimer component, polymer
component, or both components, comprise stem cells or other
cells.
32. A drug delivery composition comprising: a polymer component,
wherein the polymer component comprises a polymer having one or
more aldehyde groups; a dendrimer component, wherein the dendrimer
component comprises a dendrimer having at least 2 branches with one
or more surface groups, wherein less than 75% of the surface groups
comprise at least one primary or secondary amine; and at least one
drug combined with at least one of the polymer component and
dendrimer component.
33. The composition of claim 32, wherein the polymer and dendrimer
components are in a combination effective for the composition to
adhere to a biological tissue.
34. A method for local delivery of a drug to a biological tissue,
comprising: applying to the biological tissue the drug delivery
composition of claim 32; and permitting the at least one drug to
diffuse from the composition into the biological tissue.
35. A method for making an adhesive, the method comprising:
providing a first solution comprising a polymer component, wherein
the polymer component comprises a polymer having one or more
aldehyde groups; providing a second solution comprising a dendrimer
component, wherein the dendrimer component comprises a dendrimer
having at least 2 branches with one or more surface groups, wherein
less than 75% of the surface groups comprise at least one primary
or secondary amine; and combining the first and second solutions
together to produce an adhesive.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Patent
Application No. 61/474,984, filed Apr. 13, 2011, which is
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This disclosure relates to biocompatible adhesive materials,
such as for use with biological tissues and/or medical implants, as
well as methods and kits for making and using the biocompatible
adhesive materials.
BACKGROUND
[0003] A number of tissue adhesives have been used in various
medical procedures and applications, including topical wound
closure, supplementing or replacing surgical sutures or staples,
adhesion of synthetic materials to biological tissues, and drug
delivery. A number of known tissue adhesives, however, are
unsuitable for many applications, for example, due to toxic
degradation products, slow curing, poor mechanical strength, and
other drawbacks.
[0004] Several varieties of hydrogel adhesives have been developed,
which are nontoxic and have improved properties. These hydrogels
are generally formed by reacting a component having nucleophilic
groups with a component having electrophilic groups that react to
form a crosslinked network. However, these hydrogels typically
dissolve too quickly, lack sufficient adhesion, or have
insufficient mechanical strength.
[0005] Therefore, it would be desirable to provide improved
adhesive formulations that overcome one or more of the
above-described disadvantages.
SUMMARY
[0006] In one aspect, compositions and methods are provided for
adhering, sealing, or treating one or more biological tissues. The
method may include combining a polymer component and a dendrimer
component in any manner to form an adhesive formulation, and
contacting one or more biological tissues with the adhesive
formulation. In embodiments, the polymer component comprises a
polymer having one or more aldehyde groups. In embodiments, the
dendrimer component comprises a dendrimer having at least 2 arms or
branches with one or more surface groups. In certain embodiments,
less than 75% of the surface groups comprise at least one primary
or secondary amine. In some embodiments, the adhesive formulations
are used in a method for treating, adhering, or sealing a
biological tissue, the method comprising (1) providing a first
solution comprising a polymer component, wherein the polymer
component comprises a polymer having one or more aldehyde groups,
(2) providing a second solution comprising a dendrimer component,
wherein the dendrimer component comprises a dendrimer having at
least 2 branches with one or more surface groups, wherein less than
75% of the surface groups comprise at least one primary or
secondary amine, (3) combining the first and second solutions
together to produce an adhesive formulation and contacting one or
more biological tissues with the adhesive formulation, and (4)
allowing the adhesive formulation to cure.
[0007] In another aspect, kits are provided for making and
delivering an adhesive composition. The kit may include a first
part that comprises a polymer component and a second part that
comprises a dendrimer component. The polymer component may comprise
a polymer having one or more aldehyde groups. The dendrimer
component may comprise a dendrimer having at least 2 arms or
branches with one or more surface groups. In certain embodiments,
less than 75% of the surface groups comprise at least one primary
or secondary amine. The kit may include means for mixing the first
and second parts together.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 depicts the normalized fluorescence and images of
aldehyde-coated fluorescent microspheres (f-MS) on the surfaces of
tissues from three regions of the small intestine.
[0009] FIG. 2 depicts the interfacial fluorescence and images of a
labeled dendrimer:dextran formulation applied to the surfaces of
tissues from three regions of the small intestine.
[0010] FIG. 3 depicts the correlation between the normalized
fluorescence off-MS and labeled dendrimer:dextran formulations when
applied to the surfaces of tissues from three regions of the small
intestine.
[0011] FIG. 4 depicts the maximum load of three adhesive
formulations that contain dextran solutions of different
concentrations.
[0012] FIG. 5 depicts the maximum load at failure for different
adhesive formulations containing dextran solutions of different
concentrations.
[0013] FIG. 6 depicts the fluorescence at the interface between
various adhesive formulations and jejunum tissues.
[0014] FIG. 7 depicts the correlation between fluorescence
(surrogate for tissue amine content) and max load of adhesive
formulations applied to the jejunum.
[0015] FIG. 8 depicts the biocompatibility of several adhesive
formulations using 3T3 fibroblasts.
[0016] FIGS. 9 and 10 depict pertinent histopathological findings
for various adhesive formulations.
[0017] FIG. 11 depicts a comparison of gelation times for adhesive
formulations described herein and several adhesives containing
aminated poly(ethyleneglycol).
[0018] FIG. 12 depicts the maximum load of various adhesive
formulations containing dendrimer or PEG amine.
[0019] FIG. 13 depicts the gelation times of PEG amine adhesives of
various molecular weights and dendrimer containing adhesive
formulations.
[0020] FIG. 14 depicts the tensile test results for PEG amine
adhesives made with PEG amities of different molecular weights.
[0021] FIG. 15 depicts the distribution of the intensity of the
microspheres in healthy and diseased models.
[0022] FIG. 16 depicts the average intensity of the microspheres
applied to healthy and diseased tissues.
[0023] FIGS. 17 and 18 depicts the tissue surface coverage of
fluorescent microspheres in healthy and diseased colon tissue.
[0024] FIG. 19 depicts the fluorescent microspheres' intensity in
healthy rabbit colon, diseased rabbit colon, healthy rat colon, and
diseased rat colon.
[0025] FIGS. 20 and 21 depict histology scoring for healthy and
diseased tissues with and without incisions and with or without the
application of an adhesive formulation.
[0026] FIG. 22 depicts one embodiment of a kit containing the
components of an adhesive formulation.
DETAILED DESCRIPTION
[0027] Improved compositions and methods have been developed for
adhering, sealing, or treating one or more biological tissues.
Generally, these adhesive formulations comprise a dendrimer
component and a polymer component. In some embodiments, the
adhesive formulations are used as tissue adhesives, tissue
sealants, tissue treatments, matrix materials, fillers, coatings,
or a combination thereof.
[0028] The adhesive formulations described herein achieve better
adhesion, sealing, and/or treatment that previously known adhesives
because the dendrimer component has amines on less than 75% of its
surface groups.
[0029] As used herein, the term "adhering" generally refers to
affixing, permanently or temporarily, two or more biological
tissues, or two or more regions of a biological tissue. As used
herein, the term "sealing" generally refers to covering, at least
partially, or filling, at least partially, one or more sites on one
or more biological tissues, such as a wound. As used herein, the
term "treating" generally refers to improving the response of at
least one biological tissue to which one or more adhesive
formulations is applied. In some embodiments, the "response" that
is improved or enhanced includes inflammation, healing, or
both.
[0030] Generally, the adhesive formulations may be used on any
internal or external biological tissues. The biological tissues may
be human or other mammalian tissue. The biological tissues may be
natural or artificially generated. The biological tissues may be
skin, bone, ocular, muscular, vascular, or an internal organ, such
as lung, intestine, heart, liver, etc.
[0031] The adhesive formulation can be applied to a tissue site in
a human or other animal patient, for example, during a surgical or
other medical procedure. In one embodiment, the adhesive
formulation is used to create an anastomosis. In some embodiment,
the adhesive formulation is used to adhere, seal, and/or treat a
wound, lesion, or a combination thereof. For example, the adhesive
formulation may be applied to slow-healing or troublesome wounds,
such as those suffered by diabetics.
[0032] In one embodiment, the adhesive formulation may be used to
secure or help secure a medical implant, such as an orthopedic
implant, within a human or other animal patient.
Dendrimer Component
[0033] In one embodiment, the dendrimer component comprises a
dendrimer having amines on less than 75% of its surface groups,
which are commonly referred to as "terminal groups" or "end
groups." As used herein, the term "dendrimer" refers to any
compound with a polyvalent core covalently bonded to two or more
dendritic branches. In one embodiment, the amines are primary
amines. In another embodiment, the amines are secondary amines. In
yet another embodiment, one or more surface groups have at least
one primary and at least one secondary amine.
[0034] In one embodiment, the dendrimer extends through at least 2
generations. In another embodiment, the dendrimer extends through
at least 3 generations. In yet another embodiment, the dendrimer
extends through at least 4 generations. In still another
embodiment, the dendrimer extends through at least 5 generations.
In a further embodiment, the dendrimer extends through at least 6
generations. In still a further embodiment, the dendrimer extends
through at least 7 generations.
[0035] In one embodiment, the dendrimer may have a molecular weight
from about 1,000 to about 1,000,000 Daltons. In a further
embodiment, the dendrimer may have a molecular weight from about
3,000 to about 120,000 Daltons. In another embodiment, the
dendrimer may have a molecular weight from about 10,000 to about
100,000 Daltons. In yet another embodiment, the dendrimer may have
a molecular weight from about 20,000 to about 40,000 Daltons.
[0036] Generally, the dendrimer may be made using any known
methods. In one embodiment, the dendrimer is made by oxidizing a
starting dendrimer having surface groups comprising at least one
hydroxyl group so that at least a portion of the surface groups
comprise at least one amine. In another embodiment, the dendrimer
is made by oxidizing a starting generation 5 (G5) dendrimer having
surface groups comprising at least one hydroxyl group so that at
least a portion of the surface groups comprise at least one amine.
In yet another embodiment, the dendrimer is made by oxidizing a
starting G5 dendrimer having surface groups comprising at least one
hydroxyl group so that about 25% of the surface groups comprise at
least one amine. In a particular embodiment, the dendrimer is a G5
dendrimer having primary amines on about 25% of the dedrimer's
surface groups.
[0037] In one embodiment, the dendrimer is a
poly(amidoamine)-derived (PAMAM) dendrimer. In another embodiment,
the dendrimer is a G5 PAMAM-derived dendrimer. In yet another
embodiment, the dendrimer is a G5 PAMAM-derived dendrimer having
primary amines on about 25% of the dendrimer's surface groups.
[0038] In one embodiment, the dendrimer is a
poly(propyleneimine)-derived dendrimer.
[0039] In certain embodiments, the dendrimer component is combined
with a liquid to form a dendrimer component solution. In one
embodiment, the dendrimer component solution is an aqueous
solution. In one embodiment, the solution comprises water,
phosphate buffer saline (PBS), Dulbecco's Modified Eagle's Medium
(DMEM), or any combination thereof. In one embodiment, the
dendrimer component concentration in the dendrimer component
solution is about 5% to about 25% by weight. In another embodiment,
the dendrimer component concentration in the dendrimer component
solution is about 10% to about 20% by weight. In a further
embodiment, the dendrimer component concentration in the dendrimer
component solution is about 11% to about 15% by weight.
[0040] In some instances, the dendrimer component or dendrimer
component solution may further comprise an additive. Generally, the
amount of additive may vary depending on the application, tissue
type, concentration of the dendrimer component solution, the type
of dendrimer component, concentration of the polymer component
solutions, and/or the type of polymer component. Example of
suitable additives, include but are not limited to, pH modifiers,
thickeners, antimicrobial agents, colorants, surfactants, and
radio-opaque compounds. Specific examples of these types of
additives are described herein. In one embodiment, the dendrimer
component solution comprises a foaming additive.
[0041] In particular embodiments, the dendrimer component or
dendrimer component solution comprises at least one drug. In such
embodiments, the adhesive formulation may serve as a matrix
material for controlled release of drug. The drug may be
essentially any drug suitable for local, regional, or systemic
administration from a quantity of the adhesive formulation that has
been applied to one or more tissue sites in a patient. In one
embodiment, the drug comprises a thrombogenic agent. Non-limiting
examples of thrombogenic agents include thrombin, fibrinogen,
homocysteine, estramustine, and combinations thereof. In another
embodiment, the drug comprises an anti-inflammatory agent.
Non-limiting examples of anti-inflammatory agents include
indomethacin, salicyclic acid acetate, ibuprophen, sulindac,
piroxicam, naproxen, and combinations thereof. In still another
embodiment, the drug comprises an anti-neoplastic agent. In still
other embodiments, the drug is one for gene therapy. For example,
the drug may comprise siRNA molecules to combat cancer. Other drugs
are envisioned.
[0042] In other particular embodiments, the dendrimer component or
dendrimer component solution comprises one or more cells. For
example, the adhesive formulation may serve as a matrix material
for delivering cells to a tissue site at which the adhesive
formulation has been applied. In embodiments, the cells may
comprise endothelial cells (EC), endothelial progenitor cells
(EPC), hematopoietic stem cells, or other stem cells. In one
embodiment, the cells are capable of releasing factors to treat
cardiovascular disease and/or to reduce restenosis. Other types of
cells are envisioned.
Polymer Component
[0043] Generally, the polymer component comprises a polymer with
one or more functional groups capable of reacting with one or more
functional groups on a biological tissue and/or one or more
functional groups on the dendrimer component.
[0044] In certain embodiments, the polymer is at least one
polysaccharide. In these embodiments, the at least one
polysaccharide may be linear, branched, or have both linear and
branched sections within its structure. Generally, the at least one
polysaccharide may be natural, synthetic, or modified--for example,
by cross-linking, altering the polysaccharide's substituents, or
both. In one embodiment, the at least one polysaccharide is
plant-based. In another embodiment, the at least one polysaccharide
is animal-based. In yet another embodiment, the at least one
polysaccharide is a combination of plant-based and animal-based
polysaccharides. Non-limiting examples of polysaccharides include,
but are not limited to, dextran, chitin, starch, agar, cellulose,
hyaluronic acid, or a combination thereof.
[0045] In certain embodiments, the at least one polymer has a
molecular weight from about 1,000 to about 1,000,000 Daltons. In
one embodiment, the at least one polymer has a molecular weight
from about 5,000 to about 15,000 Daltons. Unless specified
otherwise, the "molecular weight" of the polymer refers to the
number average molecular weight.
[0046] In some embodiments, the polymer is functionalized so that
its structure includes one or more functional groups that will
react with one or more functional groups on a biological tissue
and/or one or more functional groups on the dendrimer component. In
one embodiment, the one or more functional groups incorporated into
the polymer's structure is aldehyde.
[0047] In certain embodiments, the polymer's degree of
functionalization is adjustable. The "degree of functionalization"
generally refers to the number or percentage of reactive groups on
the polymer that are replaced or converted to the desired one or
more functional groups. In one embodiment, the degree of
functionalization is adjusted based on the type of tissue to which
the adhesive is applied, the concentration(s) of the components,
and/or the type of polymer on dendrimer used in the adhesive. In
one embodiment, the degree of functionalization is from about 10%
to about 75%. In another embodiment, the degree of
functionalization is from about 15% to about 50%. In yet another
embodiment, the degree of functionalization is from about 20% to
about 30%.
[0048] In one embodiment, the polymer is dextran with a molecular
weight of about 10 kDa. In another embodiment, the polymer is
dextran having about 50% of its hydroxyl group converted to
aldehydes. In a further embodiment, the polymer is dextran with a
molecular weight of about 10 kDa and about 50% of its hydroxyl
groups converted to aldehydes.
[0049] In some embodiments, a polysaccharide is oxidized to include
a desired percentage of one or more aldehyde functional groups.
Generally, this oxidation may be conducted using any known means.
For example, suitable oxidizing agents include, but are not limited
to, periodates, hypochlorites, ozone, peroxides, hydroperoxides,
persulfates, and percarbonates. In one embodiment, the oxidation is
performed using sodium periodate. Typically, different amounts of
oxidizing agents may be used to alter the degree of
functionalization.
[0050] In certain embodiments, the polymer component is combined
with a liquid to form a polymer component solution. In one
embodiment, the polymer component solution is an aqueous solution.
In one embodiment, the solution comprises water, PBS, DMEM, or any
combination thereof.
[0051] Generally, the polymer component solution may have any
suitable concentration of polymer component. In one embodiment, the
polymer component concentration in the polymer component solution
is about 5% to about 40% by weight. In another embodiment, the
polymer component concentration in the polymer component solution
is about 5% to about 30% by weight. In yet another embodiment, the
polymer component concentration in the polymer component solution
is about 5% to about 25% by weight. Typically, the concentration
may be tailored and/or adjusted based on the particular
application, tissue type, and/or the type and concentration of
dendrimer component used.
[0052] The polymer component or polymer component solution may also
comprise one or more additives. In one embodiment, the additive is
compatible with the polymer component. In another embodiment, the
additive does not contain primary or secondary amines. Generally,
the amount of additive varies depending on the application, tissue
type, concentration of the polymer component solution, the type of
polymer component and/or dendrimer component. Examples of suitable
additives, include, but are not limited to, pH modifiers,
thickeners, antimicrobial agents, colorants, surfactants, and
radio-opaque compounds. In other embodiments, the polymer component
solution comprises a foaming agent.
[0053] In certain embodiments, the pH modifier is an acidic
compound. Examples of acidic pH modifiers include, but are not
limited to, carboxylic acids, inorganic acids, and sulfonic acids.
In other embodiments, the pH modifier is a basic compound. Examples
of basic pH modifiers include, but are not limited to, hydroxides,
alkoxides, nitrogen-containing compounds other than primary and
secondary amines, basic carbonates, and basic phosphates.
[0054] Generally, the thickener may be selected from any known
viscosity-modifying compounds, including, but not limited to,
polysaccharides and derivatives thereof, such as starch or
hydroxyethyl cellulose.
[0055] Generally, the surfactant may be any compound that lowers
the surface tension of water.
[0056] In one embodiment, the surfactant is an ionic
surfactant--for example, sodium lauryl sulfate. In another
embodiment, the surfactant is a neutral surfactant. Examples of
neutral surfactants include, but are not limited to,
polyoxyethylene ethers, polyoxyethylene esters, and polyoxyethylene
sorbitan.
[0057] In one embodiment, the radio-opaque compound is barium
sulfate, gold particles, or a combination thereof.
[0058] In particular embodiments, the polymer component or polymer
component solution comprises at least one drug. In such
embodiments, the adhesive formulation may serve as a matrix
material for controlled release of drug. The drug may be
essentially any drug suitable for local, regional, or systemic
administration from a quantity of the adhesive formulation that has
been applied to one or more tissue sites in a patient. In one
embodiment, the drug comprises a thrombogenic agent. Non-limiting
examples of thrombogenic agents include thrombin, fibrinogen,
homocysteine, estramustine, and combinations thereof. In another
embodiment, the drug comprises an anti-inflammatory agent.
Non-limiting examples of anti-inflammatory agents include
indomethacin, salicyclic acid acetate, ibuprophen, sulindac,
piroxicam, naproxen, and combinations thereof. In still another
embodiment, the drug comprises an anti-neoplastic agent. In still
other embodiments, the drug is one for gene or cell therapy. For
example, the drug may comprise siRNA molecules to combat cancer.
Other drugs are envisioned.
[0059] In other particular embodiments, the polymer component or
polymer component solution comprises one or more cells. For
example, the adhesive formulation may serve as a matrix material
for delivering cells to a tissue site at which the adhesive
formulation has been applied. In embodiments, the cells may
comprise endothelial cells (EC), endothelial progenitor cells
(EPC), hematopoietic stem cells, or other stem cells. In one
embodiment, the cells are capable of releasing factors to treat
cardiovascular disease and/or to reduce restenosis. Other types of
cells are envisioned.
Adhesive Formulation
[0060] Generally, the adhesive formulations described herein may be
formed by combining the polymer component or polymer component
solution, and the dendrimer component or dendrimer component
solution in any manner. In some embodiments, the polymer component
or polymer component solution, and the dendrimer component or
dendrimer component solution are combined before contacting a
biological tissue with the adhesive formulation. In other
embodiments, the polymer component or polymer component solution,
and the dendrimer component or dendrimer component solution are
combined, in any order, on a biological tissue. In further
embodiments, the polymer component or polymer component solution is
applied to a first biological tissue, the dendrimer component or
dendrimer component solution is applied to a second biological
tissue, and the first and second biological tissues are contacted.
In still a further embodiment, the polymer component or polymer
component solution is applied to a first region a biological
tissue, the dendrimer component or dendrimer component solution is
applied to a second region of a biological tissue, and the first
and second regions are contacted.
[0061] Generally, the adhesive formulation may be applied to one or
more biological tissues as an adhesive, sealant, and/or treatment.
The one or more biological tissues may be diseased or healthy. In
one embodiment, the adhesive formulation is applied to one or more
biological tissues as an adhesive. In another embodiment, the
adhesive formulation is applied to one or more biological tissues
as a sealant. In a further embodiment, the adhesive formulation is
applied to one or more biological tissues as a treatment. In an
additional embodiment, the adhesive formulation is applied to one
or more biological tissues as an adhesive and sealant. In still
another embodiment, the adhesive formulation is applied to one or
more biological tissues as an adhesive and treatment. In yet
another embodiment, the adhesive formulation is applied to one or
more biological tissues as a sealant and treatment. In a still
further embodiment, the adhesive formulation is applied to one or
more biological tissues as an adhesive, sealant, and treatment.
[0062] As used herein, the adhesive formulation is a "treatment"
when it improves the response of at least one biological tissue to
which it is applied. In some embodiments, the improved response is
lessening overall inflammation, improving the specific response at
the wound site/interface of the tissue and adhesive formulation,
enhancing healing, or a combination thereof. As used herein, the
phrase "lessening overall inflammation" refers to an improvement of
histology scores that reflect the severity of inflammation. As used
herein, the phrase "improving the specific response at the wound
site/interface of the tissue and adhesive formulation" refers to an
improvement of histology scores that reflect the severity of
serosal neutrophils. As used herein, the phrase "enhancing healing"
refers to an improvement of histology scores that reflect the
severity of serosal fibrosis.
[0063] After contacting one or more biological tissues, the
adhesive formulations may be allowed adequate time to cure or gel.
When the adhesive formulation "cures" or "gels," as those terms are
used herein, it means that the reactive groups on the polymer
component, dendrimer component, and one or more biological tissues
have undergone one or more reactions. Not wishing to be bound by
any particular theory, it is believed that the adhesive
formulations described herein are effective because the polymer
component reacts with both the dendrimer component and the surface
of the biological tissues. In certain embodiments, the polymer
component's aldehyde functional groups react with the amines on the
dendrimer component and the biological tissues to form imine bonds.
In these embodiments, it is believed that the amines on the
dendrimer component react with a high percentage of the aldehydes
on the polymer component, thereby reducing toxicity and increasing
biocompatibility of the adhesive formulations. Typically, the time
needed to cure or gel the adhesive formulations will vary based on
a number of factors, including, but not limited to, the
characteristics of the polymer component and/or dendrimer
component, the concentrations of the polymer component solution
and/or the dendrimer component solution, and the characteristics of
the one or more biological tissues. In embodiments, the adhesive
formulation will cure sufficiently to provide desired bonding or
sealing shortly after the components are combined. The gelation or
cure time should provide that a mixture of the components can be
delivered in fluid form to a target area before becoming too
viscous or solidified and then once applied to the target area sets
up rapidly thereafter. In one embodiment, the gelation or cure time
is less than 120 seconds. In another embodiment, the gelation or
cure time is between 3 and 60 seconds. In a particular embodiment,
the gelation or cure time is between 5 and 30 seconds.
[0064] In certain embodiments, one or more foaming agents are added
to the polymer component solution and/or the dendrimer component
solution before the solutions are combined. In one embodiment, the
foaming agents comprise a two part liquid system comprising Part 1
and Part 2, wherein Part 1 comprises a bicarbonate and Part 2
comprises an aqueous solution of di- or polyaldehydes and a
titrant. A wide range of di- or polyaldhydes exist, and their
usefulness is restricted largely by availability and by their
solubility in water. For example, aqueous glyoxal (ethanedial) is
useful, as is aqueous glutaraldehyde (pentadial). Water soluble
mixtures of di- and polyaldehydes prepared by oxidative cleavage of
appropriate carbohydrates with periodate, ozone or the like may
also be useful.
[0065] A titrant is most preferably employed in the liquid solution
of Part 2. More specifically, the titrant is an organic or
inorganic acid, buffer, salt, or salt solution which is capable of
reacting with the bicarbonate component of Part 1 to generate
carbon dioxide and water as reaction by-products. The carbon
dioxide gas that is generated creates a foam-like structure of the
adhesive formulation and also causes the volume of the adhesive
formulation to expand.
[0066] Most preferably, the titrant is an inorganic or organic acid
that is present in an amount to impart an acidic pH to the
resulting mixture of the Part 1 and Part 2 components. Preferred
acids that may be employed in the practice of the present invention
include phosphoric acid, sulfuric acid, hydrochloric acid, acetic
acid, and citric acid.
Tissue Specific Formulations
[0067] Generally, the polymer component and the dendrimer component
that are combined to form the adhesive formulation may be tailored
for specific biological tissues. For example, the type of
components or the amounts of one or both of the components may be
adjusted. Not wishing to be bound by any particular theory, it is
believed that performing an analysis to determine the density of
amine groups on the surface of a biological tissue may guide the
determination of how to alter the adhesive formulations. In one
embodiment, aldehyde-coated fluorescent microspheres (f-MS) are
applied to various tissues to aid this analysis.
[0068] Generally, the adhesive formulations may be adjusted in any
manner to compensate for differences between tissues. In one
embodiment, the amount of polymer component is increased or
decreased while the amount of dendrimer component is unchanged. In
another embodiment, the amount of dendrimer component is increased
or decreased while the amount of polymer component is unchanged. In
another embodiment, the concentration of the polymer component
solution is increased or decreased while the dendrimer component or
dendrimer component solution is unchanged. In yet another
embodiment, the concentration of the dendrimer component solution
is increased or decreased while the polymer component or polymer
component solution is unchanged. In a further embodiment, the
concentrations of the both the polymer component solution and the
dendrimer component solution are changed.
[0069] When the amine density on the surface of a particular
biological tissue is unknown due to disease, injury, or otherwise,
an excess of polymer component or polymer component solution may,
in some embodiments, be added when the adhesive formulation is
first applied, then the amount of polymer component or polymer
component solution may be reduced, e.g., incrementally or
drastically, until the desired effect is achieved. The "desired
effect," in this embodiment, may be an appropriate or adequate
curing time, adhesion, sealing, or a combination thereof. Not
wishing to be bound by any particular theory, it is believed that
an excess of polymer component or polymer component solution may be
required, in some instances, to obtain the desired effect when the
amine density on a biological tissue is low. Therefore, adding an
excess will help the user, in this embodiment, achieve adequate
sealing or adhesion in less time. This is particularly desirable in
emergency situations.
[0070] In other embodiments, however, a lower amount of polymer
component or polymer component solution may be added when the
adhesive formulation is first applied, then the amount of polymer
component or polymer component solution may be increased, e.g.,
incrementally or drastically, until the desired effect is achieved,
which may be adequate curing time, adhesion, sealing, or a
combination thereof.
Adhesive Formulation Kits
[0071] In another aspect, a kit is provided that comprises a first
part that includes a polymer component or polymer component
solution, and a second part that includes a dendrimer component or
dendrimer component solution. The kit may further include an
applicator or other device means, such as a multi-compartment
syringe, for storing, combining, and delivering the two parts
and/or the resulting adhesive formulation to a tissue site.
[0072] In one embodiment, the kit comprises separate reservoirs for
the polymer component solution and the dendrimer component
solution. In certain embodiments, the kit comprises reservoirs for
polymer component solutions of different concentrations. In other
embodiments, the kit comprises reservoirs for dendrimer component
solutions of different concentrations.
[0073] In one embodiment, the kit comprises instructions for
selecting an appropriate concentration or amount of at least one of
the polymer component, polymer component solution, dendrimer
component, or dendrimer component solution to compensate or account
for at least one characteristic of one or more biological tissues.
In one embodiment, the adhesive formulation is selected based on
one or more predetermined tissue characteristics. For example,
previous tests, such as those described herein, may be performed to
determine the number of density of bonding groups on a biological
tissue in both healthy and diseased states. Alternatively, a rapid
tissue test may be performed to assess the number or density of
bonding groups. Quantification of tissue bonding groups can be
performed by contacting a tissue with one or more materials that
(1) have at least one functional group that specifically interacts
with the bonding groups, and (2) can be assessed by way of
fluorescence or detachment force required to separate the bonding
groups and the material. In another embodiment, when the density of
bonding groups on a biological tissue is unknown, an excess of the
polymer component, such as one containing aldehydes, may be
initially added as described herein to gauge the density of bonding
groups on the surface of the biological tissue.
[0074] In certain embodiments, the kit comprises at least one
syringe. In one embodiment, the syringe comprises separate
reservoirs for the polymer component solution and the dendrimer
component solution. The syringe may also comprise a mixing tip that
combines the two solutions as the plunger is depressed. The mixing
tip may be releasably securable to the syringe (to enable exchange
of mixing tips), and the mixing tip may comprise a static mixer. In
some embodiments, the reservoirs in the syringe may have different
sizes or accommodate different volumes of solution. In other
embodiments, the reservoirs in the syringe may be the same size or
accommodate the same volumes of the solution. In a further
embodiment, one reservoir may comprise Part 1 of the foaming
composition described hereinabove, and a second reservoir may
comprise Part 2 of the foaming composition.
[0075] FIG. 22 depicts one embodiment of a syringe 220. The syringe
220 includes a body 221 with two reservoirs (223, 224). A dendrimer
component solution is disposed in the first reservoir 223, and a
polymer component solution is disposed in the second reservoir 224.
The two reservoirs (223, 224) are emptied by depressing the plunger
222, which pushes the contents of the two reservoirs (223, 224)
into the mixing tip 225 and out of the syringe 220.
[0076] In a further embodiment, one or more of the reservoirs of
the syringe may be removable. In this embodiment, the removable
reservoir may be replaced with a reservoir containing a polymer
component solution or a dendrimer component solution of a desired
concentration.
[0077] In a preferred embodiment, the kit is sterile. For example,
the components of the kit may be packaged together, for example in
a tray, pouch, and/or box. The packaged kit may be sterilized using
known techniques such as electron beam irradiation, gamma
irradiation, ethylene oxide sterilization, or other suitable
techniques.
EXAMPLES
[0078] The present invention is further illustrated by the
following examples, which are not to be construed in any way as
imposing limitations upon the scope thereof. On the contrary, it is
to be clearly understood that resort may be had to various other
aspects, embodiments, modifications, and equivalents thereof which,
after reading the description herein, may suggest themselves to one
of ordinary skill in the art without departing from the spirit of
the present invention or the scope of the appended claims. Thus,
other aspects of this invention will be apparent to those skilled
in the art from consideration of the specification and practice of
the invention disclosed herein.
Example 1
Preliminary Testing of Various Tissues
[0079] The conjugation of aldehyde-coated fluorescent microspheres
(f-MS) was used to probe tissue-surface chemistry, and provide a
mechanistic basis for the variability in adhesive mechanics. This
technique is described in Artzi, N., et al. ADV. MATER. 21, 2009,
1-5.
[0080] The technique is used because tissue-tissue interfacial
stress is regulated in nature with variations in surface chemistry.
Examining these changes helped explain tissue-adhesive formulation
interactions, and assisted the development tissue-specific adhesive
formulations.
[0081] In this example, tissues from three regions of the small
intestine were tested: jejunum, duodenum, and ileum. These tissues
were selected because chemical differences drive gastrointestinal
(GI) tract physiology. The spectrum of contractility and
persitalsis, pH, and surface chemistry across the GI bed allow for
a profound modulation of nutrition, inflammation, infection, etc.
Biopsies of each tissue were prepared with equal surface area (20
mm.sup.2) and submerged in 0.5 mL of 0.5% f-MS solutions for 20
minutes on a rocker at 37.degree. C. The tissue samples then were
thoroughly rinsed with 10 mL PBS three times. Following rinsing,
the fluorescent intensity at the surface of the tissue samples was
measured. Images were obtained with a fluorescence microscope to
confirm the presence of the f-MS. A fluoroscein isothiocyanate
(FITC) filter was used for fluorescein and tetramethylrhodamine
isothiocyanate (TRITC) for propidium iodide (tissue staining).
[0082] The normalized fluorescence of the f-MS on the surface of
each tissue sample is shown in FIG. 1. The tissues from the three
regions of the small intestine had surfaces with different numbers
of amine groups, as demonstrated by the variable fluorescent
intensity of the conjugated f-MS. In this figure, a p-value
<0.05 was considered to denote statistical significance.
Example 2
Interfacial Fluorescence of a Biocompatible Adhesive
[0083] The information concerning natural GI surface chemistry in
Example 1 was used to create adhesive formulations that interacted
differentially with specific tissues to create adhesive
formulations that are more effective GI wall sealants. Leakage of
gut content is a frequent surgical complication that can result in
local infection and systemic sepsis, peritonitis, and often the
need for reoperation. Adhesive formulations were created by
matching adhesive formulation and tissue properties. This example
determined that differences in tissue surfaces--in this case, amine
density in duodenum, jejunum, and ileum--affect interactions with
adhesive formulations of varied aldehyde content and densities.
[0084] To characterize the adhesive formulation's morphology at the
tissue-adhesive interface, the dendrimer component was labeled with
fluorescein. This technique is described in Artzi, N., et al. ADV.
MATER. 21, 2009, 1-5. The dendrimer used in this example was a G5
dendrimer with 25% of its surface groups having primary amines
instead of hydroxyl groups. The dendrimer had a molecular weight of
about 30 kDaltons.
[0085] The dendrimer was dissolved in 6 mL dimethylsulfoxide,
followed by the addition of 0.030 g of
6-(fluorescein-5-carboxyamido)hexanoic acid (Invitrogen, Carlsbad,
Calif. 92008) and 12 .mu.L triethylamine. The mixture was stirred
at room temperature for 48 h. The resulting solid after solvent
evaporation was dissolved in 100 mL water, dialyzed, and
lyophilized. The fluorescein-labeled dendrimer was then added to
water to create a solution having 1% of the total solid content
(12.5%) by weight of the fluorescein-labeled dendrimer
(G5-25-12.5)
[0086] A separate aqueous solution of dextran was created. The
dextran, in this example, had a molecular weight of about 10
kDalton, and 50% of its hydroxyl groups had been converted to
aldehydes. The aqueous solution contained 11.25% by weight of the
dextran (D10-50-11.25).
[0087] The two solutions G5-25-12.5 and D10-50-11.25 were then
combined and applied to separate samples of three tissues from
different regions of the small intestine: jejunum, duodenum, and
ileum. The combination of solutions was allowed to cure for 5
min.
[0088] The tissue samples were then cryosectioned (20 .mu.m
sections) and stained with propidium iodide. The adhesive's
morphology at the tissue:adhesive interface was quantified using
image analysis techniques (Leica Microsystems, MetaMorph.RTM.) to
characterize the transitory material between tissue surface and
material bulk. FIG. 2 depicts the interfacial fluorescence of the
labeled dendrimer. The differences in normalized fluorescence
demonstrated that the labeled dendrimer:dextran (formulation
G5-25-12.5:D10-50-11.25) had different reactivity with the three
tissue surfaces due to the different amine densities presented by
these tissues. In this figure, a p-value <0.05 was considered to
denote statistical significance.
[0089] To demonstrate that the f-MS analysis accurately predicts
the interaction of a particular tissue with the labeled
dendrimer:dextran (formulation G5-25-12.5:D10-50-11.25), the
normalized fluorescence of the f-MS and the normalized fluorescence
of the labeled dendrimer were plotted and compared as shown in FIG.
3. In this figure, a linear correlation exists between the
normalized fluorescence of the labeled dendrimer (X-axis) and the
f-MS. As a result, the f-MS was used as a tool to accurately gauge
the behavior of various adhesive compositions when applied to
different types of tissue. The information obtained from the f-MS
can be used to determine how the dendrimer component, polymer
component, and/or solution concentrations should be adjusted to
compensate for the varying characteristics of different tissue
types. These data supported the notion that the functional groups
on tissues and in the adhesive formulations influence
aldehyde-mediated adhesive interactions, providing a functional
basis for tissue-specific adhesive design. In this example, the
microspheres assay correlated with mechanical quantification of
adhesion strength measured in the tensile strength tests described
herein.
Example 3
Effect of Polymer Component Solution Concentration on Adhesion
Strength
[0090] The adhesion strength following the application of a
biocompatible adhesive to various tissues was measured with
monotonic uniaxial tensile testing (Bose.RTM. Biodynamic Test
Instrument, Minnetonka, Minn., USA). In this example, three
adhesive formulations were applied to three tissues from the small
intestine. The three adhesive formulations contained the same
dendrimer component solution: the G5-25-12.5 solution described in
Example 2. Each polymer component solution contained various
concentrations of a dextran (D10-50) with a molecular weight of 10
kDaltons, and 50% of its hydroxyl groups converted to aldehydes.
The first polymer component solution had a dextran concentration of
25% by weight (D10-50-25), the second polymer component solution
had a dextran concentration of 15% by weight (D10-50-15), and the
third polymer component solution had a dextran concentration of
11.25% by weight (D10-50-11.25).
[0091] Each individual dextran solution was combined with an equal
volume of the dendrimer solution to create three formulations: (1)
D10-50-25:G5-25-12.5, (2) D10-50-15:G5-25-12.5, and (3)
D10-50-11.25:G5-25-12.5. Adhesive test elements were created by
evenly distributing 200 .mu.L of these adhesive formulations
between two uniformly sized tissue biopsies (discs of 8 mm
diameter, total test element thickness of 1 mm). All three
formulations were tested on three tissues from various regions of
the small intestine: ileum, jejunum, and duodenum. The tissue
surfaces were gently dried prior to applying the adhesive
formulation.
[0092] After applying each formulation between tissue surfaces and
allowing 5 minutes for curing, the adhesive test elements were
displaced at a constant rate of 0.05 mm/s, and the load response
was continuously recorded (200 measurements/s). Recorded loads were
normalized to account for adhesive test element cross-sectional
area. The max load (N) of each formulation on each tissue type is
shown in FIG. 4. On all three tissues, increasing the concentration
of the dextran solution increased the maximum load. Therefore,
altering the concentration of the polymer component solution--in
this example, a dextran-containing solution--can compensate for the
different structures and chemical makeup of various types of
tissues. In this figure, a p-value <0.05 was considered to
denote statistical significance.
[0093] The max load (N) of three additional formulations and their
adhesion to the jejunum was also tested. Like the above-described
formulations, each contained dendrimer component solutions of the
same concentration--in this case, G5-25,12.5. However, different
concentrations of polymer component solutions were used to make
each formulation: 7.5% by weight, 9.375% by weight, and 20% by
weight. Therefore, the three additional formulations were (4)
D10-50-7.5:G5-25-12.5, (5) D10-50-9.375:G5-25-12.5, and (6)
D10-50-20:G5-25-12.5.
[0094] The max loads (N) of these three additional formulations and
the previously-described formulations are shown in FIG. 5. As the
concentration of the polymer component solution increased, the max
load (N) increased. This result further illustrated how adjusting
the concentration of the polymer component solution can be used to
compensate for the differences between tissues. In this figure, a
p-value <0.05 was considered to denote statistical significance.
Images were obtained of the adhesive formulations tested in FIG. 5
after being applied to the jejunum tissues. The images were
obtained by tagging 1% of the dendrimer with
6-(fluorescein-5-carboxyamido)hexanoic acid before applying the
formulation to the jejunum. The jejunum was then frozen with liquid
nitrogen and maintained at -80.degree. C. overnight. The tissue was
then cryosectioned (20 .mu.m sections) and stained with propidium
iodide. Fluorescence microscopy images were taken using FITC
(material, fluorescein) and TRITC (tissue, propidium iodide)
filters. The images revealed the interface between the tissues and
the adhesive formulations.
[0095] The fluorescence at the interface between the jejunum and
the adhesive formulations of FIGS. 5 was also measured. As shown in
FIG. 6, the normalized fluorescence increased as the concentration
of polymer component solution--in this case, a dextran
solution--increased. In addition to correlating with the
concentration of the polymer component solution, the normalized
fluorescence also correlated with the max load of the adhesive
formulations applied to the jejunum. This relationship is
illustrated in FIG. 7. These relationships showed how the
analytical techniques described herein can be used to predict and
estimate the changes that should be made to the adhesive
formulations to compensate for a number of variables, including the
characteristics of different tissue types. These tests also allow
for determining how the state of a disease and its severity can
alter the number and functionality of chemical groups on tissue
surfaces.
Example 4
Biocompatibility of Various Adhesive Formulations
[0096] The biocompatibility of three of the adhesive formulations
described in Example 3 were tested using 3T3 fibroblasts. The three
adhesive formulations were (a) D10-50-7.5:G5-25-12.5, (b)
D10-50-15:G5-25-12.5, and (c) D10-50-25:G5-25-12.5. The cell
survival was measured after 1 day, 1 week, and 1 month.
[0097] Adhesive disks of 5 mm diameter were kept in 5 mL of media
at 37.degree. C. for 1 day, 1 week, and 1 month. At each interval,
100K 3T3 fibroblasts were incubated for 24 hours in 1 mL of media
containing the degradation products of the disks. After 24 hours,
cell death was measured using CytotoxONE Membrane Integrity Assay
(Promega Corp., Madison, Wis., USA), and the number of dead cells
was normalized to the total number of both live and dead cells. The
number of live cells was determined by trypsinizing and counting
the cells. As shown in FIG. 8, the biocompatibility test showed
very high cell survival. Images of the adhesive formulations
implanted into the tissue of mice also showed good
biocompatibility.
[0098] Similarly, FIGS. 9 and 10 depict pertinent histopathological
findings of various adhesive formulations. In this test, 15 .mu.m
tissue sections were stained with Hematoxylin and Eosin for
semi-quantitative scoring (i.e., 1=minimal, 2=mild, 3=severe) of
pertinent parameters (e.g., inflammation, fibrosis, giant cells
etc.) for each dose group by time point.
Example 5
Comparison of Gelation Times for Various Adhesive Formulations
[0099] The gelation times for several adhesive formulations
described herein were compared to each other, and to adhesives made
using polyethylene glycol (PEG) amine and dextran. The gelation
time in this example is the time required for the two components to
form an adhesive hydrogel, which is indicative of crosslinking
density and stiffness.
[0100] Six different adhesive formulations, as described herein,
were produced for this test. Each adhesive formulation consisted of
combination of G5-25-20 (which is a 20% by weight aqueous solution
of a G5 dendrimer with 25% of its surface groups having primary
amines instead of hydroxyl groups) and various concentrations of an
aqueous solution of D10-50, which is a 10 kDalton dextran with 50%
of its hydroxyl groups replaced with aldehydes. The concentrations,
by weight percent, of the D10-50 solutions were 4, 5, 7.5, 10, 15,
and 25. As shown in FIG. 11, the gelation times for the adhesive
compositions with polymer component solution concentrations of 10,
15, and 25% by weight were very similar, and much shorter than the
gelation times for the adhesive compositions with polymer component
solution concentrations of 4, 5, and 7.5% by weight.
[0101] FIG. 11 also includes the gelation times of various
adhesives containing PEG amine and dextran (D10-50). The PEG amine
in this example was an eight-armed polyether with primary amines at
the end of each arm, and a molecular weight of 10 kDalton. A 6.67%
by weight aqueous solution of PEG-amine--referred to in this
example as P8-10-6.67--was combined with dextran solutions of the
following concentrations: 4, 5, 10, 15, and 25% by weight. The
gelation times of these six PEG adhesives are plotted in FIG. 11.
For comparison purposes, all of the adhesive formulations compared
in FIG. 11 were formulated so that each had the same number of
either PEG amine molecules or dendrimer molecules.
[0102] As shown in FIG. 11, the gelation times of the
dendrimer-containing adhesive formulations were faster than those
of the PEG amine adhesives at every concentration of dextran. Not
wishing to be bound by any particular theory, this discrepancy may
be caused by the lower steric hindrance of the dendrimer component
of the adhesive formulations described herein; therefore, the
reaction between the dendrimer component and the dextran is more
likely to occur than the reaction between PEG amine and
dextran.
[0103] The maximum load of the adhesives of FIG. 11 are plotted in
FIG. 12. With the exception of the adhesive formulations containing
a 5% by weight dextran solution, the dendrimer-containing adhesive
formulations had a higher maximum load.
Example 6
Comparison of Adhesive Formulations Containing Dendrimers with
Those Containing PEG Amine of Different Molecular Weights
[0104] In Example 5, the dendrimer component was a dendrimer with
primary amines on 25% of its surface groups instead of hydroxyl
groups, and a molecular weight of 30 kDalton. In contrast, the
PEG-amine used in the other adhesive formulations of Example 5 had
a molecular weight of 10 kDalton. To test the effects of the
molecular weight of the PEG amine, a series of adhesives containing
PEG-amines with different molecular weights were produced. Although
the molecular weights differed, each adhesive contained the same
number of PEG amine molecules.
[0105] Specifically, three different PEG amine components were used
to formulate fifteen adhesives by combining the three PEG amine
components with dextran solutions having concentrations of 5, 7.5,
10, 15, and 25% by weight. The three different PEG amine components
each had eight arms (P8) and consisted of a 6.67% by weight
solution of a 10 kDalton PEG-amine, a 13.34% by weight solution of
a 20 kDalton PEG amine, and a 26.68% by weight solution of a 40
kDalton PEG amine.
[0106] FIG. 13 depicts the gelation times of these fifteen
adhesives along with the gelation times for adhesive formulations
made by combining each dextran solution with a 20% solution of a 30
kDalton G5 dendrimer having primary amines on 25% of its surface
groups instead of hydroxyl groups. As the molecular weight of the
PEG in the PEG amine adhesive increased, the gelation time of the
PEG amine adhesive became more similar to the dendrimer-containing
adhesive formulations described herein. The 20 kDalton and 40
kDalton PEG amine adhesives had approximately the same gelation
times, which were less than the gelation time for the 10 kDalton
PEG amine. Not wishing to be bound by any particular theory, this
difference is believed to be caused by the differences in steric
hindrance: the PEG amines with higher molecular weights are less
sterically hindered and, as a result, more likely to react with the
other component.
[0107] Although the 20 kDalton and 40 kDalton PEG amine adhesives
had faster gelling times than the 10 kDalton PEG amine adhesive,
the maximum load handled by the 10 kDalton PEG amine adhesive was
higher than maximum loads of the 20 kDalton and 40 kDalton PEG
amine adhesives. Not wishing to be bound by any particular theory,
this may be related to the competition in the reaction between the
aldehydes in the dextran with the amines in the tissue and the
amines in the PEG amine. FIG. 14 depicts these results.
Example 7
Disease Model--Colitis Induction
[0108] A disease model was designed based on colitis. To develop
the model, the differences in amine density of healthy and diseased
colon was studied. The experiments indicated whether the surface of
the tissue was modified by inducing inflammation, and to what
extent it affected adhesion and cohesion strengths of the
dendrimer-containing adhesive formulation.
[0109] For the diseased model, dinitrobenzene sulfonic acid (DNBS)
was instilled at a concentration of 80 mg/mL. After 24 hours, the
rabbits were euthanized and the colon was harvested, cleaned, and
incubated for 20 minutes in a 0.5% aldehyde coated microspheres
solution. The tissues were frozen, cryo-sectioned, and stained with
propidium iodide. The intensity of the microspheres was quantified
by fluorescence microscopy.
[0110] Fluorescence microscope images showed that the healthy model
had a higher amine density and higher distribution along the
surface. Hematoxylin and eosin stains (H & E) of the
cryosectioned healthy and diseased tissues demonstrated alteration
in the morphology of the luminal surface, as evidenced by
hyperplasia and thickening of the colon with cell infiltrates.
Serosal layer chemistry was impacted as well, as evidenced by
alteration of the f-MS intensity and continuity throughout the
colon in the diseased state, i.e., there were less amine groups
present at the surface. FIG. 15 shows the distribution of the
microsphere's intensity in the healthy and diseased models. FIG. 16
shows the average intensity of the microspheres applied to healthy
and diseased tissues (colitis). This graph shows that the
microsphere's average fluorescence intensity, and hence the
adhesion of the dendrimer containing adhesive formulations to the
tissue, dramatically dropped (over 60%) when the tissue was
inflamed.
[0111] In this model, the reduction in amine density affected the
interaction between the tissue and adhesive formulation, and
adhesion strength. More specifically, in this particular example,
colitis resulted in lower f-MS conjugation to tissues, as evidenced
by lower fluorescence intensity at the tissue surface and lower
surface coverage, as shown in FIG. 17. The lower surface coverage,
in this example, occurred in isolated areas and perturbed the
continuous surface coverage observed in the healthy state.
Therefore, the concentrations of the component solutions or the
amounts of the components administered may be adjusted to
compensate for this characteristic of diseased tissue. Specific
material formulations can be designed to address the
disease-induced alteration of tissue surface chemistry.
Specifically, in certain embodiments, a formulation containing a
higher amount or concentration of polymer component can be
administered to compensate for the decreased number of amines on
the surface of diseased tissue, thereby improving the reaction
yield, improving adhesion, or both.
Example 8
Disease Model--Cancer
[0112] The analyses in Example 7 also were performed on cancerous
tissue from the colon of rats. In the cancer model, the diseased
tissue had a higher f-MS tissue surface coverage than the healthy
tissue, as shown in FIG. 18. The opposite results produced by the
colitis model (Example 7) and the cancer model indicated the
importance of tuning the adhesive formulation as described herein
to compensate for disease-induced tissue alteration. A more
thorough comparison of the f-MS intensity differences in the
colitis model (Example 7) and the cancer model is shown in FIG. 19.
FIG. 19 shows that the inflamed colon tissue from the colitis model
(Example 7) had a lower tissue surface coverage of f-MS compared to
the cancerous tissue in the cancer model.
Example 9
Tissue Response to Adhesive Formulation--Colitis Model
[0113] The overall tissue response to an adhesive formulation was
determined by collecting a series of histology scores that
indicated the severity of inflammation, serosal heterophils (at the
interface with the material indicating response to the material),
and serosal fibrosis (which indicates healing). The adhesive
formulation used in this example included D10-50-15 and G5-25-20.
The histology scores were collected for the 6 colon tissue groups
listed in Table 1. In each of the groups containing incisions, the
incisions were sutured. Photomicrographs of H & E stained
sections were also collected, and explained in Table 1. The tissue
samples were collected in a manner similar to the process of
Example 7.
TABLE-US-00001 TABLE 1 Colon Tissue Groups Group No. Description
Photomicrograph I Healthy control group - Colon appeared normal. no
incision, no adhesive formulation II Colitis control group -
Colonic mucosa was diffusely necrotic with an intense no incision,
no heterophilic inflammatory response. adhesive formulation III
Healthy colon - with Mild increase in the number of inflammatory
cells in the incision, no adhesive mucosa; moderate heterophilic
inflammatory response, formulation predominantly in association
with the suture material; mild inflammation and fibrosis was seen
along serosal surface in association with the incision site/suture
material. IV Colitis colon - with Colonic mucosa was diffusely
necrotic with an intense incision, no adhesive heterophilic
inflammatory response that extended from formulation the mucosal
surface, surrounded the suture material, and infiltrated deep to
the serosal surface, where there was associated fibrosis. V Healthy
colon - with Mild inflammation and fibrosis was at the interface
incision, with between the serosa and adhesive material. adhesive
formulation VI Colitis colon - with A small portion of relatively
normal mucosa was visible; incision, with the remainder of the
colonic wall was necrotic with an adhesive formulation intense
heterophilic inflammatory response that extended from the mucosal
surface, surrounded the suture material, and infiltrated deep to
the serosa to surround the adhesive material.
[0114] FIG. 20 shows the histology scores collected from the
tissues from Groups I-VI. In the healthy tissues (Groups I, III,
and V), inflammation in Group V was less than the inflammation in
Group III. Therefore, in this test, the inflammation of healthy
tissues with an incision was reduced upon adhesive application. In
the healthy tissues, the healing responses were similar whether or
not the adhesive formulation was applied, and there was no evidence
of any adverse response to the adhesive formulation.
[0115] In the diseased tissues (Groups II, IV, and V), the overall
inflammation was higher than in the healthy tissues. It was
observed, however, that application of the adhesive formulation
increased the inflammatory response at the interface between the
tissue and the adhesive formulation. Moreover, in this particular
test, application of the adhesive formulation to the diseased
tissues increased serosal fibrosis--i.e., enhanced healing of the
tissues. Both serosal heterophils and serosal fibrosis were
affected by the state of the tissue (diseased or healthy), which
further demonstrated the importance, in certain instances, of
tuning the adhesive formulations in view of specific
microenvironmental conditions.
Example 10
Tissue Response to Adhesive Formulation--Cancer Model
[0116] The analyses in Example 9 also were performed on cancerous
tissues. As in Example 9, the adhesive formulation used in this
example included D10-50-15 and G5-25-20. The overall tissue
response to the adhesive formulation was determined by collecting a
series of histology scores that indicated the severity of
inflammation, serosal heterophils (at the interface with the
material indicating response to the material), and serosal fibrosis
(which indicates healing). The histology scores were collected for
the 6 tissue groups listed in Table 2. In each of the groups
containing incisions, the incisions were sutured. Photomicrographs
of H & E stained sections were also collected, and explained in
Table 2. The tissue samples were collected in a manner similar to
the process of Example 8.
TABLE-US-00002 TABLE 2 Cancer Tissue Groups Group No. Description
Photomicrograph VII. Healthy control group - Colon appeared normal.
no incision, no adhesive formulation VIII. Cancer control group -
Dysplastic epithelium, characterized by nuclear crowding no
incision, no and hyperchromatism, indicated tumor site. adhesive
formulation IX. Healthy colon - with Mild increase in the number of
inflammatory cells in the incision, no adhesive mucosa; mild
inflammation and fibrosis was seen along formulation the serosal
surface in association with the incision site/suture material. X.
Cancer colon - with Colonic mucosa showed a heterophilic
inflammatory incision, no adhesive response surrounding the suture
material, and infiltrates formulation to the serosal surface, where
there was associated fibrosis. XI. Healthy colon - with Coalescing
mucosal inflammation and fibrosis at the incision, with interface
between the serosa and adhesive material was adhesive formulation
observed, especially next to the suture material. XII. Cancer colon
- with A small portion of relatively normal mucosa was visible,
incision, with nuclear crowding showed tumor formation; the
adhesive formulation remainder of the colonic wall showed a mixture
of neutrophilic inflammatory response and fibrous tissue.
[0117] FIG. 21 shows the histology scores collected from the
tissues from Groups VII-XII. The overall inflammation and surface
inflammation were similar in healthy (Groups VII, IX, and XI) and
diseased (Groups VIII, X, and XII) states. Similar to the colitis
model (Example 8), the addition of adhesive formulation imparted
healing in the cancerous tissues, as demonstrated by the elevation
of surface heterophils.
* * * * *